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1.
Nat Commun ; 12(1): 847, 2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33558503

RESUMO

A large G4C2-repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Neuronal degeneration associated with this expansion arises from a loss of C9orf72 protein, the accumulation of RNA foci, the expression of dipeptide repeat (DPR) proteins, or all these factors. We report the discovery of a new targeting sequence that is common to all C9orf72 transcripts but enables preferential knockdown of repeat-containing transcripts in multiple cellular models and C9BAC transgenic mice. We optimize stereopure oligonucleotides that act through this site, and we demonstrate that their preferential activity depends on both backbone stereochemistry and asymmetric wing design. In mice, stereopure oligonucleotides produce durable depletion of pathogenic signatures without disrupting protein expression. These oligonucleotides selectively protect motor neurons harboring C9orf72-expansion mutation from glutamate-induced toxicity. We hypothesize that targeting C9orf72 with stereopure oligonucleotides may be a viable therapeutic approach for the treatment of C9orf72-associated neurodegenerative disorders.


Assuntos
Proteína C9orf72/genética , Expansão das Repetições de DNA/genética , Mutação/genética , Oligonucleotídeos/química , Oligonucleotídeos/genética , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Animais , Proteína C9orf72/química , Éxons/genética , Glutamatos/toxicidade , Íntrons/genética , Camundongos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Processamento de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estereoisomerismo
2.
Neurology ; 96(9): e1369-e1382, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33495376

RESUMO

OBJECTIVE: To delineate the full phenotypic spectrum, discriminative features, piloting longitudinal progression data, and sample size calculations of replication factor complex subunit 1 (RFC1) repeat expansions, recently identified as causing cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS). METHODS: Multimodal RFC1 repeat screening (PCR, Southern blot, whole-exome/genome sequencing-based approaches) combined with cross-sectional and longitudinal deep phenotyping in (1) cross-European cohort A (70 families) with ≥2 features of CANVAS or ataxia with chronic cough (ACC) and (2) Turkish cohort B (105 families) with unselected late-onset ataxia. RESULTS: Prevalence of RFC1 disease was 67% in cohort A, 14% in unselected cohort B, 68% in clinical CANVAS, and 100% in ACC. RFC1 disease was also identified in Western and Eastern Asian individuals and even by whole-exome sequencing. Visual compensation, sensory symptoms, and cough were strong positive discriminative predictors (>90%) against RFC1-negative patients. The phenotype across 70 RFC1-positive patients was mostly multisystemic (69%), including dysautonomia (62%) and bradykinesia (28%) (overlap with cerebellar-type multiple system atrophy [MSA-C]), postural instability (49%), slow vertical saccades (17%), and chorea or dystonia (11%). Ataxia progression was ≈1.3 Scale for the Assessment and Rating of Ataxia points per year (32 cross-sectional, 17 longitudinal assessments, follow-up ≤9 years [mean 3.1 years]) but also included early falls, variable nonlinear phases of MSA-C-like progression (SARA points 2.5-5.5 per year), and premature death. Treatment trials require 330 (1-year trial) and 132 (2-year trial) patients in total to detect 50% reduced progression. CONCLUSIONS: RFC1 disease is frequent and occurs across continents, with CANVAS and ACC as highly diagnostic phenotypes yet as variable, overlapping clusters along a continuous multisystemic disease spectrum, including MSA-C-overlap. Our natural history data help to inform future RFC1 treatment trials. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that RFC1 repeat expansions are associated with CANVAS and ACC.


Assuntos
Atrofia de Múltiplos Sistemas/diagnóstico , Atrofia de Múltiplos Sistemas/genética , Proteína de Replicação C/genética , Adulto , Idoso , Ataxia , Vestibulopatia Bilateral , Estudos de Coortes , Expansão das Repetições de DNA , Progressão da Doença , Europa (Continente) , Exoma , Feminino , Testes Genéticos , Humanos , Imagem por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Atrofia de Múltiplos Sistemas/diagnóstico por imagem , Fenótipo , Valor Preditivo dos Testes , Turquia , Doenças Vestibulares
3.
Nature ; 589(7841): 246-250, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33442040

RESUMO

Autism spectrum disorder (ASD) is an early-onset developmental disorder characterized by deficits in communication and social interaction and restrictive or repetitive behaviours1,2. Family studies demonstrate that ASD has a substantial genetic basis with contributions both from inherited and de novo variants3,4. It has been estimated that de novo mutations may contribute to 30% of all simplex cases, in which only a single child is affected per family5. Tandem repeats (TRs), defined here as sequences of 1 to 20 base pairs in size repeated consecutively, comprise one of the major sources of de novo mutations in humans6. TR expansions are implicated in dozens of neurological and psychiatric disorders7. Yet, de novo TR mutations have not been characterized on a genome-wide scale, and their contribution to ASD remains unexplored. Here we develop new bioinformatics methods for identifying and prioritizing de novo TR mutations from sequencing data and perform a genome-wide characterization of de novo TR mutations in ASD-affected probands and unaffected siblings. We infer specific mutation events and their precise changes in repeat number, and primarily focus on more prevalent stepwise copy number changes rather than large expansions. Our results demonstrate a significant genome-wide excess of TR mutations in ASD probands. Mutations in probands tend to be larger, enriched in fetal brain regulatory regions, and are predicted to be more evolutionarily deleterious. Overall, our results highlight the importance of considering repeat variants in future studies of de novo mutations.


Assuntos
Transtorno do Espectro Autista/genética , Expansão das Repetições de DNA/genética , Predisposição Genética para Doença , Adolescente , Adulto , Transtorno do Espectro Autista/patologia , Encéfalo/metabolismo , Criança , Variações do Número de Cópias de DNA/genética , Feminino , Feto/metabolismo , Mutação em Linhagem Germinativa/genética , Humanos , Análise dos Mínimos Quadrados , Masculino , Pessoa de Meia-Idade , Idade Paterna , Adulto Jovem
4.
Adv Exp Med Biol ; 1281: 67-76, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33433869

RESUMO

The identification of C9orf72 gene has led to important scientific progresses and has considerably changed our clinical practice. However, a decade after C9orf72 discovery, some important clinical questions remain unsolved. The reliable cutoff for the pathogenic repeat number and the implication of intermediate alleles in frontotemporal dementia, amyotrophic lateral sclerosis, or in other diseases are still uncertain. The occurrence of an anticipation phenomenon - at the clinical and molecular levels - in C9orf72 kindreds is still debated as well, and the factors driving age at onset and phenotype variability are largely unknown. All these questions have a significant impact not only in clinical practice for diagnosis and genetic counseling but also in a research context for the initiation of therapeutic trials. In this chapter, we will address all those issues and summarize the recent updates about clinical aspects of C9orf72 disease, focusing on both the common and the less typical phenotypes.


Assuntos
Esclerose Amiotrófica Lateral , Demência Frontotemporal , Doença de Pick , Esclerose Amiotrófica Lateral/diagnóstico , Esclerose Amiotrófica Lateral/genética , Proteína C9orf72/genética , Expansão das Repetições de DNA/genética , Demência Frontotemporal/diagnóstico , Demência Frontotemporal/genética , Humanos , Proteínas/genética
5.
Proc Natl Acad Sci U S A ; 117(40): 25104-25115, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32958650

RESUMO

Maintaining the fidelity of nascent peptide chain (NP) synthesis is essential for proteome integrity and cellular health. Ribosome-associated quality control (RQC) serves to resolve stalled translation, during which untemplated Ala/Thr residues are added C terminally to stalled peptide, as shown during C-terminal Ala and Thr addition (CAT-tailing) in yeast. The mechanism and biological effects of CAT-tailing-like activity in metazoans remain unclear. Here we show that CAT-tailing-like modification of poly(GR), a dipeptide repeat derived from amyotrophic lateral sclerosis with frontotemporal dementia (ALS/FTD)-associated GGGGCC (G4C2) repeat expansion in C9ORF72, contributes to disease. We find that poly(GR) can act as a mitochondria-targeting signal, causing some poly(GR) to be cotranslationally imported into mitochondria. However, poly(GR) translation on mitochondrial surface is frequently stalled, triggering RQC and CAT-tailing-like C-terminal extension (CTE). CTE promotes poly(GR) stabilization, aggregation, and toxicity. Our genetic studies in Drosophila uncovered an important role of the mitochondrial protease YME1L in clearing poly(GR), revealing mitochondria as major sites of poly(GR) metabolism. Moreover, the mitochondria-associated noncanonical Notch signaling pathway impinges on the RQC machinery to restrain poly(GR) accumulation, at least in part through the AKT/VCP axis. The conserved actions of YME1L and noncanonical Notch signaling in animal models and patient cells support their fundamental involvement in ALS/FTD.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Esclerose Amiotrófica Lateral/genética , Proteína C9orf72/genética , Proteínas de Drosophila/genética , Demência Frontotemporal/genética , Metaloendopeptidases/genética , Proteínas Mitocondriais/genética , Proteoma/genética , Receptores Notch/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Arginina/genética , Expansão das Repetições de DNA/genética , Modelos Animais de Doenças , Drosophila melanogaster/genética , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Células HEK293 , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Biossíntese de Proteínas , Ribossomos/genética , Ribossomos/metabolismo , Transdução de Sinais/genética
7.
Am J Hum Genet ; 107(3): 445-460, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32750315

RESUMO

Tandem repeats are proposed to contribute to human-specific traits, and more than 40 tandem repeat expansions are known to cause neurological disease. Here, we characterize a human-specific 69 bp variable number tandem repeat (VNTR) in the last intron of WDR7, which exhibits striking variability in both copy number and nucleotide composition, as revealed by long-read sequencing. In addition, greater repeat copy number is significantly enriched in three independent cohorts of individuals with sporadic amyotrophic lateral sclerosis (ALS). Each unit of the repeat forms a stem-loop structure with the potential to produce microRNAs, and the repeat RNA can aggregate when expressed in cells. We leveraged its remarkable sequence variability to align the repeat in 288 samples and uncover its mechanism of expansion. We found that the repeat expands in the 3'-5' direction, in groups of repeat units divisible by two. The expansion patterns we observed were consistent with duplication events, and a replication error called template switching. We also observed that the VNTR is expanded in both Denisovan and Neanderthal genomes but is fixed at one copy or fewer in non-human primates. Evaluating the repeat in 1000 Genomes Project samples reveals that some repeat segments are solely present or absent in certain geographic populations. The large size of the repeat unit in this VNTR, along with our multiplexed sequencing strategy, provides an unprecedented opportunity to study mechanisms of repeat expansion, and a framework for evaluating the roles of VNTRs in human evolution and disease.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Esclerose Amiotrófica Lateral/genética , Evolução Molecular , Sequências de Repetição em Tandem/genética , Idoso , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Esclerose Amiotrófica Lateral/patologia , Expansão das Repetições de DNA/genética , Feminino , Regulação da Expressão Gênica/genética , Humanos , Masculino , Repetições Minissatélites/genética , Fenótipo , Especificidade da Espécie
9.
Nat Commun ; 11(1): 3354, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620797

RESUMO

Expansion of an intronic (GGGGCC)n repeat region within the C9orf72 gene is a main cause of familial amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). A hallmark of c9ALS/FTD is the accumulation of misprocessed RNAs, which are often targets of cellular RNA surveillance. Here, we show that RNA decay mechanisms involving upstream frameshift 1 (UPF1), including nonsense-mediated decay (NMD), are inhibited in c9ALS/FTD brains and in cultured cells expressing either of two arginine-rich dipeptide repeats (R-DPRs), poly(GR) and poly(PR). Mechanistically, although R-DPRs cause the recruitment of UPF1 to stress granules, stress granule formation is independent of NMD inhibition. Instead, NMD inhibition is primarily a result from global translational repression caused by R-DPRs. Overexpression of UPF1, but none of its NMD-deficient mutants, enhanced the survival of neurons treated by R-DPRs, suggesting that R-DPRs cause neurotoxicity in part by inhibiting cellular RNA surveillance.


Assuntos
Esclerose Amiotrófica Lateral/genética , Proteína C9orf72/genética , Demência Frontotemporal/genética , Degradação do RNAm Mediada por Códon sem Sentido , RNA Helicases/metabolismo , Transativadores/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Expansão das Repetições de DNA , Conjuntos de Dados como Assunto , Embrião de Mamíferos , Feminino , Lobo Frontal/patologia , Demência Frontotemporal/patologia , Humanos , Íntrons/genética , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Cultura Primária de Células , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA-Seq , Transativadores/genética
11.
J Vis Exp ; (158)2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32420986

RESUMO

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington's disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.


Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , Expansão das Repetições de DNA , Neurônios/patologia , Iniciação Traducional da Cadeia Peptídica , Fragmentos de Peptídeos/toxicidade , RNA Antissenso/genética , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Neurônios/efeitos dos fármacos
12.
Cancer Sci ; 111(7): 2472-2481, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32391628

RESUMO

In this phase II multicenter study (JALSG AML209-FLT3-SCT), we aimed to prospectively elucidate the role of allogeneic hematopoietic stem cell transplantation (allo-HSCT) at first complete remission (CR1) for FLT3-internal tandem duplication (ITD)-positive AML. Newly diagnosed de novo AML patients with FLT3-ITD were enrolled at the achievement of CR1 and received allo-HSCT as soon as possible after the first consolidation therapy. Mutations of 57 genes in AML cells at diagnosis were also analyzed. Among 48 eligible patients with a median age of 38.5 (17-49) years, 36 (75%) received allo-HSCT at a median of 108 days after CR1. The median follow-up was 1726 days. The primary end-point, 3-year disease-free survival (DFS) based on an intent to treat analysis, was 43.8% (95% confidence interval [CI], 30%-57%), suggesting the efficacy of this treatment because the lower limit of the 95% CI exceeded the threshold response rate of 20%. The 3-year overall survival, post-transplant DFS, and non-relapse mortality rates were 54.2% (95% CI, 39%-67%), 58.3% (95% CI, 41%-72%), and 25.0% (95% CI, 12%-40%), respectively. The median ITD allelic ratio (AR) was 0.344 (0.006-4.099). Neither FLT3-ITD AR nor cooccurring genetic alterations was associated with a poor DFS. This prospective study indicated the efficacy and safety of allo-HSCT for FLT3-ITD AML patients in CR1. This study was registered at: www.umin.ac.jp/ctr/ as #UMIN000003433.


Assuntos
Expansão das Repetições de DNA , Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Tirosina Quinase 3 Semelhante a fms/genética , Adolescente , Adulto , Terapia Combinada , Feminino , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Indução de Remissão , Transplante Homólogo , Resultado do Tratamento , Adulto Jovem
13.
Neuron ; 107(2): 292-305.e6, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32375063

RESUMO

GGGGCC hexanucleotide repeat expansions (HREs) in C9orf72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) and lead to the production of aggregating dipeptide repeat proteins (DPRs) via repeat associated non-AUG (RAN) translation. Here, we show the similar intronic GGCCTG HREs that causes spinocerebellar ataxia type 36 (SCA36) is also translated into DPRs, including poly(GP) and poly(PR). We demonstrate that poly(GP) is more abundant in SCA36 compared to c9ALS/FTD patient tissue due to canonical AUG-mediated translation from intron-retained GGCCTG repeat RNAs. However, the frequency of the antisense RAN translation product poly(PR) is comparable between c9ALS/FTD and SCA36 patient samples. Interestingly, in SCA36 patient tissue, poly(GP) exists as a soluble species, and no TDP-43 pathology is present. We show that aggregate-prone chimeric DPR (cDPR) species underlie the divergent DPR pathology between c9ALS/FTD and SCA36. These findings reveal key differences in translation, solubility, and protein aggregation of DPRs between c9ALS/FTD and SCA36.


Assuntos
Esclerose Amiotrófica Lateral/genética , Proteína C9orf72/genética , Dipeptídeos/genética , Demência Frontotemporal/genética , Proteínas Mutantes Quiméricas/genética , Ataxias Espinocerebelares/genética , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Elementos Antissenso (Genética)/genética , Expansão das Repetições de DNA , Feminino , Humanos , Íntrons/genética , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Sequências Repetitivas de Ácido Nucleico
14.
Nat Neurosci ; 23(5): 615-624, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32284607

RESUMO

Hexanucleotide expansions in C9orf72, which encodes a predicted guanine exchange factor, are the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although repeat expansion has been established to generate toxic products, mRNAs encoding the C9ORF72 protein are also reduced in affected individuals. In this study, we tested how C9ORF72 protein levels affected repeat-mediated toxicity. In somatic transgenic mice expressing 66 GGGGCC repeats, inactivation of one or both endogenous C9orf72 alleles provoked or accelerated, respectively, early death. In mice expressing a C9orf72 transgene with 450 repeats that did not encode the C9ORF72 protein, inactivation of one or both endogenous C9orf72 alleles exacerbated cognitive deficits, hippocampal neuron loss, glial activation and accumulation of dipeptide-repeat proteins from translation of repeat-containing RNAs. Reduced C9ORF72 was shown to suppress repeat-mediated elevation in autophagy. These efforts support a disease mechanism in ALS/FTD resulting from reduced C9ORF72, which can lead to autophagy deficits, synergizing with repeat-dependent gain of toxicity.


Assuntos
Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Animais , Expansão das Repetições de DNA/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
16.
Nat Commun ; 11(1): 2022, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332745

RESUMO

The thymus is a primary lymphoid organ that plays an essential role in T lymphocyte maturation and selection during development of one arm of the mammalian adaptive immune response. Although transcriptional mechanisms have been well documented in thymocyte development, co-/post-transcriptional modifications are also important but have received less attention. Here we demonstrate that the RNA alternative splicing factor MBNL1, which is sequestered in nuclear RNA foci by C(C)UG microsatellite expansions in myotonic dystrophy (DM), is essential for normal thymus development and function. Mbnl1 129S1 knockout mice develop postnatal thymic hyperplasia with thymocyte accumulation. Transcriptome analysis indicates numerous gene expression and RNA mis-splicing events, including transcription factors from the TCF/LEF family. CNBP, the gene containing an intronic CCTG microsatellite expansion in DM type 2 (DM2), is coordinately expressed with MBNL1 in the developing thymus and DM2 CCTG expansions induce similar transcriptome alterations in DM2 blood, which thus serve as disease-specific biomarkers.


Assuntos
Proteínas de Ligação a DNA/genética , Distrofia Miotônica/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Timo/crescimento & desenvolvimento , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Expansão das Repetições de DNA , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Íntrons/genética , Masculino , Camundongos , Camundongos Knockout , Repetições de Microssatélites/genética , Pessoa de Meia-Idade , Distrofia Miotônica/sangue , Distrofia Miotônica/imunologia , Processamento de RNA/imunologia , RNA-Seq , Timo/imunologia , Adulto Jovem
17.
Hum Genet ; 139(10): 1233-1246, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32277284

RESUMO

Approximately 3% of the human genome is composed of short tandem repeat (STR) DNA sequence known as microsatellites, which can be found in both coding and non-coding regions. When associated with genic regions, expansion of microsatellite repeats beyond a critical threshold causes dozens of neurological repeat expansion disorders. To better understand the molecular pathology of repeat expansion disorders, precise cloning of microsatellite repeat sequence and expansion size is highly valuable. Unfortunately, cloning repeat expansions is often challenging and presents a significant bottleneck to practical investigation. Here, we describe a clear method for seamless and systematic cloning of practically any microsatellite repeat expansion. We use cloning and expansion of GGGGCC repeats, which are the leading genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), as an example. We employ a recursive directional ligation (RDL) technique to build multiple GGGGCC repeat-containing vectors. We describe methods to validate repeat expansion cloning, including diagnostic restriction digestion, PCR across the repeat, and next-generation long-read MinION nanopore sequencing. Validated cloning of microsatellite repeats beyond the critical expansion threshold can facilitate step-by-step characterization of disease mechanisms at the cellular and molecular level.


Assuntos
Esclerose Amiotrófica Lateral/genética , Proteína C9orf72/genética , Clonagem Molecular/métodos , Expansão das Repetições de DNA , Demência Frontotemporal/genética , Repetições de Microssatélites , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Sequência de Bases , Proteína C9orf72/metabolismo , Enzimas de Restrição do DNA/química , Escherichia coli/genética , Escherichia coli/metabolismo , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Genoma Humano , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Reação em Cadeia da Polimerase/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
18.
Ann Neurol ; 88(1): 113-122, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32285980

RESUMO

OBJECTIVE: C9orf72 expansion is the most common genetic cause of frontotemporal dementia (FTD). We examined aging trajectories of cortical thickness (CTh) and surface area in C9orf72 expansion adult carriers compared to healthy controls to characterize preclinical cerebral changes leading to symptoms. METHODS: Data were obtained from the Genetic Frontotemporal Dementia Initiative. T1-weighted magnetic resonance imaging scans were processed with CIVET 2.1 to extract vertex-wide CTh and cortical surface area (CSA). Symptomatic and presymptomatic subjects were compared to age-matched controls using mixed-effects models, controlling for demographic variables. Aging trajectories were compared between carriers and noncarriers by testing the "age by genetic status" interaction. False discovery rate corrections were applied to all vertex-wide analyses. RESULTS: The sample included 640 scans from 386 subjects, including 54 symptomatic C9orf72 carriers (72.2% behavioral variant FTD), 83 asymptomatic carriers, and 249 controls (age range = 18-86 years). Symptomatic carriers showed fairly symmetric reduction in CTh/CSA in most of the frontal lobes, in addition to large temporoparietal areas. Presymptomatic subjects had reduced CTh/CSA in more restricted areas of the medial frontoparietal lobes, in addition to scattered lateral frontal, parietal, and temporal areas. These differences were explained by faster cortical thinning linearly throughout adulthood in a similar anatomical distribution, with differences emerging in the early 30s. CSA reduction was also faster in mutation carriers predominantly in the ventrofrontal regions. INTERPRETATION: C9orf72 mutation carriers have faster cortical thinning and surface loss throughout adulthood in regions that show atrophy in symptomatic subjects. This suggests that the pathogenic effects of the mutation lead to structural cerebral changes decades prior to symptoms. ANN NEUROL 2020 ANN NEUROL 2020;88:113-122.


Assuntos
Proteína C9orf72/genética , Córtex Cerebral/diagnóstico por imagem , Expansão das Repetições de DNA , Demência Frontotemporal/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Atrofia/diagnóstico por imagem , Atrofia/genética , Feminino , Demência Frontotemporal/diagnóstico por imagem , Humanos , Imagem por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Adulto Jovem
19.
J Neurol Neurosurg Psychiatry ; 91(5): 503-511, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32132225

RESUMO

OBJECTIVES: The hexanucleotide repeat expansion in the C9orf72 gene is the most common mutation associated with amyotrophic lateral sclerosis (C9-ALS) and frontotemporal dementia (C9-FTD). Until now, it is unknown which factors define whether C9orf72 mutation carriers develop ALS or FTD. Our aim was to identify protein biomarker candidates in the cerebrospinal fluid (CSF) which differentiate between C9-ALS and C9-FTD and might be indicative for the outcome of the mutation. METHODS: We compared the CSF proteome of 16 C9-ALS and 8 C9-FTD patients and 11 asymptomatic C9orf72 mutation carriers (CAR) by isobaric tags for relative and absolute quantitation. Eleven biomarker candidates were selected from the pool of differentially regulated proteins for further validation by multiple reaction monitoring and single-molecule array in a larger cohort (n=156). RESULTS: In total, 2095 CSF proteins were identified and 236 proteins were significantly different in C9-ALS versus C9-FTD including neurofilament medium polypeptide (NEFM) and chitotriosidase-1 (CHIT1). Eight candidates were successfully validated including significantly increased ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) levels in C9-ALS compared with C9-FTD and controls and decreased neuronal pentraxin receptor (NPTXR) levels in C9-FTD versus CAR. CONCLUSIONS: This study presents a deep proteomic CSF analysis of C9-ALS versus C9-FTD patients. As a proof of concept, we observed higher NEFM and CHIT1 CSF levels in C9-ALS. In addition, we also show clear upregulation of UCHL1 in C9-ALS and downregulation of NPTXR in C9-FTD. Significant differences in UCHL1 CSF levels may explain diverging ubiquitination and autophagy processes and NPTXR levels might reflect different synapses organisation processes.


Assuntos
Esclerose Amiotrófica Lateral/líquido cefalorraquidiano , Proteína C9orf72/genética , Expansão das Repetições de DNA/genética , Demência Frontotemporal/líquido cefalorraquidiano , Adulto , Idoso , Esclerose Amiotrófica Lateral/genética , Biomarcadores/líquido cefalorraquidiano , Feminino , Demência Frontotemporal/genética , Hexosaminidases/líquido cefalorraquidiano , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Neurofilamentos/líquido cefalorraquidiano , Proteoma/análise , Imagem Individual de Molécula
20.
PLoS One ; 15(3): e0228789, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32160188

RESUMO

Large expansions of microsatellite DNA cause several neurological diseases. In Spinocerebellar ataxia type 10 (SCA10), the repeat interruptions change disease phenotype; an (ATTCC)n or a (ATCCT)n/(ATCCC)n interruption within the (ATTCT)n repeat is associated with the robust phenotype of ataxia and epilepsy while mostly pure (ATTCT)n may have reduced penetrance. Large repeat expansions of SCA10, and many other microsatellite expansions, can exceed 10,000 base pairs (bp) in size. Conventional next generation sequencing (NGS) technologies are ineffective in determining internal sequence contents or size of these expanded repeats. Using repeat primed PCR (RP-PCR) in conjunction with a high-sensitivity pulsed-field capillary electrophoresis fragment analyzer (FEMTO-Pulse, Agilent, Santa Clara, CA) (RP-FEMTO hereafter), we successfully determined sequence content of large expansion repeats in genomic DNA of SCA10 patients and transformed yeast artificial chromosomes containing SCA10 repeats. This RP-FEMTO is a simple and economical methodology which could complement emerging NGS for very long sequence reads such as Single Molecule, Real-Time (SMRT) and nanopore sequencing technologies.


Assuntos
Ataxina-10/genética , Eletroforese Capilar/métodos , Repetições de Microssatélites/genética , Ataxias Espinocerebelares/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Expansão das Repetições de DNA/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo
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